CN106854652A - A kind of willow PtCYP85A3 genes and application - Google Patents

Abstract

The invention discloses a poplar PtCYP85A3 gene and its application. The gene can improve the biomass, fruit yield and salt and drought tolerance of plants. The present invention introduces the PtCYP85A3 gene into tomatoes and poplars to illustrate the role of the PtCYP85A3 gene in It plays an important role in controlling the growth and stress resistance of tomato and poplar, and has important application value in plant genetic engineering and genetic improvement genes.

Description

A kind of poplar PtCYP85A3 gene and its application

technical field

The invention belongs to the field of biotechnology, and relates to a poplar PtCYP85A3 gene and its application in improving plant biomass, fruit yield and enhancing plant stress resistance.

Background technique

Poplar is one of the important tree species in the world. It is widely distributed all over the world. It has the characteristics of rapid growth and excellent material. It can provide board and papermaking raw materials. Genetic manipulation of poplar is also relatively easy, so scientists study it as a model woody plant (Taylor G, 2002). Poplar is rich in lignocellulose and perennial, and it is also an ideal raw material tree species for biomass energy. Not long ago, the genome sequencing of poplar has been completed (Tuskan GA et al, 2006). Scientists plan to transform poplar on this basis to make it grow faster and have a smaller canopy. , New species with lower lignin content, which is more conducive to the production of cellulosic ethanol.

The research on BRs began 40 years ago when Mitchell et al extracted a substance from rapeseed flowers that could elongate the second internode of soybean (Mitchell et al., 1970). After a series of analyzes of BRs synthetic mutants and BRs insensitive mutants, the synthesis pathway of BRs has been solved. The synthesis of plant sterol hormones is different from the synthesis of animal hormones. The precursor of animal sterol hormones is cholesterol, while the synthesis of plant brassinosteroids is based on brassinosterol. The divergence in the synthetic pathways of zoosterol hormones and phytosterol hormones begins with the formation of cyclosterols, which is a unique reaction in plants. The synthesis of brassinosteroid can be divided into two steps, the first step is the synthesis of brassinosterol, the precursor of brassinosterol, and the second step is to synthesize brassinosterol from brassinosterol.

Many mutants with defects in brassinosteroid (BR) synthesis have been discovered, such as det2 (Li et al., 1996; Fujioka et al., 1997), dwf4 (Choe et al., 1998), quasi Arabidopsis cpd (Szekeres et al., 1996), tomato dwarf (Lycopersicon esculentum; Bishop et al., 1999), pea lkb (Pisum sativum; Nomura et al., 1997, 1999), etc., the study found that the lack of BR will shrink Elongation of aerial parts, reduced fecundity, delayed senescence, altered plant vasculature and affected photomorphogenesis. The abnormal phenotype of these mutants can be restored by exogenously applying BRs. It is now widely recognized that BRs are essential chemical signals and plant hormones, and their endogenous levels are very important for maintaining normal plant growth and development.

As the sixth major plant hormone, brassinolide widely affects the growth and development of plants, especially promoting the growth of aboveground parts of plants. Recent studies have found a significant relationship between BRs and crop yield.

Rice mutants d11 and d2-1 are mutants in the BRs synthesis pathway, and d61-1 is a BRs insensitive mutant. D11 encodes a cytochrome P450 protein, which is involved in the supply of 6-deoxytycarosterol and cattailol; d61-1 is a mutant of rice BRs receptor; D2 encodes a P450 protein, which is involved in the post-C6 pathway of BRs synthesis. The d11 mutant showed a significant decrease in rice seed length, but the same width as the wild type, while the d61-1, d2 seeds were also smaller than the wild type, but their length and width were changed. From d11, d61-1, d2-1 mutants have changed the size of the seeds, indicating that BRs have a certain relationship with the size of the seeds, and the size of the seeds affects the yield of crops, so it shows that BRs may be related to the yield of crops (Tanabe et al., 2005).

DWF4 encodes the hydroxylase at position 22 of brassinosteroid, which is a key gene controlling the synthesis of BRs. There have been many reports to overexpress DWF4 in plants to increase the content of BRs in plants. For example, overexpression of AtDWF4 in Arabidopsis resulted in longer hypocotyls in overexpressed lines than in wild-type under both light and dark conditions. At maturity, the inflorescence height of the transgenic overexpressed plants was 35% of that of the wild type, the total number of siliques and branches was twice that of the wild type, and the number of seeds was 59% of that of the wild type (Choe et al., 2001). Overexpression of the DWF4 gene of maize and Arabidopsis in rice can increase the tiller number and seed yield of rice (Wu et al., 2008). Overexpression of Arabidopsis DWF4 in tobacco resulted in tobacco inflorescence heights 14% of wild-type (Choe et al., 2001).

DWF1 is the upstream gene of brassinosteroid synthesis, and the study of overexpression of DWF1 has been involved in rice and Arabidopsis. Overexpression of DWF1 in rice leads to taller plants, larger bending of internodes, more protoconicles, and elongation of the second internode (ZhiHong et al.2005). However, overexpression of AtDWF1 in Arabidopsis has no obvious phenotype (Klahre et al., 1998). Overexpression of AtDWF2 in Arabidopsis promotes vegetative growth of plants (Kim et al., 2010).

However, there is no report on whether the poplar PtCYP85A3 gene involved in the present invention can be overexpressed in plants to increase the content of BRs in plants, thereby increasing the biomass, fruit yield and stress resistance of plants.

Contents of the invention

The object of the present invention is to provide a poplar PtCYP85A3 gene and its application, by increasing the content of endogenous BRs, the biomass (such as plant height and stem diameter), fruit yield, salt and drought tolerance, etc. of the plant can be improved.

To achieve the above object, the present invention takes the following technical solutions:

A poplar PtCYP85A3 gene, the gene is the nucleotide sequence shown in SEQ ID NO.1.

The protein encoded by the above PtCYP85A3 gene, the protein is the amino acid sequence shown in SEQ ID No.2.

Among them, the full length of the PtCYP85A3 gene is 3274bp, which contains 9 exons and 8 introns, and the full length of the cDNA is 1553bp, which includes a 72bp 5' non-coding region and a 86bp 3' non-coding region. The length of the coding region of the gene is 1395bp, encoding a protein of 464 amino acids, hydrophobic amino acids account for 43.7%, the molecular weight of the protein is 53.65kD, and the isoelectric point is 9.46.

The most important purpose of the present invention is to provide the application of poplar PtCYP85A3 gene, including improving the fruit yield of plants and improving its stress resistance, and can also improve the wood biomass of woody plants. The present invention is verified by specific experiments in poplar and tomato In order to greatly increase the biomass, fruit yield and enhance the stress resistance of poplar and tomato. Wherein said stress resistance includes salt tolerance and drought resistance, etc., but the scope of protection herein is not limited to poplar and tomato, and can play the same role in all plants.

The aforementioned synthetase genes and related genes all belong to the protection scope of the present invention.

Expression vectors, recombinant vectors, recombinant strains or transgenic cell lines containing the above genes are all within the protection scope of the present invention.

The cloned gene comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can be expressed in a foreign host through a suitable expression system to obtain corresponding enzymes or other higher biological activities or yields.

The polypeptide comprising the amino acid sequence or at least part of the sequence provided by the present invention may still have biological activity or even have new biological activity after removing or substituting certain amino acids, or increase the yield or optimize protein dynamics characteristics or other efforts to obtained properties.

Genes comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can be expressed in heterologous hosts and their functions in the host metabolic chain can be understood by DNA chip technology.

The protein encoded by the nucleotide sequence provided by the present invention and the nucleotide sequence and protein functionally identical or similar to PtCYP85A3 can be synthesized.

The gene comprising the nucleotide sequence provided by the present invention or at least part of the nucleotide sequence can be constructed by genetic recombination to construct a recombinant plasmid to obtain a new biosynthetic pathway, and can also be inserted, replaced, deleted or inactivated to obtain a new biosynthetic way.

The non-ribosomal peptide synthetase provided by the invention may be derived from one or more non-ribosomal peptide synthetase domains, modules or genes by deletion, insertion or inactivation from the same or different non-ribosomal peptide synthetase systems And produce new polypeptide compounds.

Fragments or genes comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can be used to construct a non-ribosomal peptide synthetase library or a non-ribosomal peptide synthetase derived library or combined library.

The gene can also be used in genetic engineering, protein expression, enzyme catalyzed reaction, etc., and can also be used to find and discover compounds or genes used in medicine, industry or agriculture to expand the source range of the PtCYP85A3 gene, and has high application prospects.

The present invention has the following advantages:

The invention introduces the PtCYP85A3 gene into tomato and poplar, and finds that the biomass (such as plant height and stem diameter), fruit yield and salt and drought tolerance of tomato and poplar can be significantly improved. It shows that the PtCYP85A3 gene plays an important role in controlling the growth and development and stress resistance of tomato and poplar, and has important application value in the field of genetic engineering of forest trees and clonal forestry, and this gene can be applied to poplar or tomato plants genetic improvement.

Description of drawings

Fig. 1 is the amino acid sequence alignment result and phylogenetic tree of poplar and tomato; Fig. 1a is the amino acid sequence alignment result of poplar and tomato, and Fig. 1b is the phylogenetic tree.

Figure 2 shows the expression of PtCYP85A3 in different tissues of Populus trichocarpa; (a) RT-PCR and (b) Real-time PCR results of PtCYP85A3 expression in different tissues. Poplar EF1β gene was used as internal standard, in the figure, Ap (terminal bud), JL (young leaf), ML (mature leaf), Pe (petiole), EP (phloem of elongated stem), EX (xylem of elongated stem ), TP (thickened stem phloem), TX (thickened stem xylem), R (root).

Fig. 3 is the effect of different treatment time and treatment concentration on the expression level of PtCYP85A3.

Figure 4 shows the situation of PtCYP85A3 protein localization in the endoplasmic reticulum; in the figure, (a) poplar mesophyll cell protoplasts transiently express pA7-YFP, PtCYP85A3-YFP, ER-YFP, pA7-YFP is a negative control, and ER-YFP is Positive control; (b) ER-CFP and PtCYP85A3-YFP or pA7-YFP were co-transfected into poplar mesophyll cell protoplasts, and the fluorescence signal of CFP was observed at 458 nm, and the scale bar = 5 μm.

Fig. 5 is the situation of PtCYP85A3 complementary tomato d ^x mutant; in the figure, (a) vector model diagram; (bd) wild type, mutant and 35S::PtCYP85A3 phenotype, 1 bar = 5cm; b is T0 generation transgenic plants, c and d are 6-week-old and 8-week-old T1 transgenic lines, respectively.

Figure 6 is the molecular detection and phenotype of the transgenic tomato overexpressing poplar PtCYP85A3; in the figure, (a) vector schematic diagram; (b) PCR detection result of PtCYP85A3 transgenic tomato; (c) RT-PCR detection result of PtCYP85A3 transgenic tomato (d) Plant height phenotype of PtCYP85A3 transgenic tomato at 2 months, where WT means wild type, V means empty vector, L1, L2, L5 and L6 mean four independent transgenic lines.

Figure 7 is the molecular detection results of PtCYP85A3 transgenic poplar. In the figure, (a) schematic diagram of transformation vector; (b) PCR detection result of transgenic poplar; (c) GUS staining result of transgenic poplar; (d) RT-PCR detection result of transgenic poplar.

Fig. 8 is the plant height and diameter phenotype of PtCYP85A3 transgenic poplar; Among the figure, (a) the plant height phenotype of the transgenic poplar of normal growth 9 weeks in the greenhouse, 1bar=20cm; (b) the normal growth of 13 weeks in the greenhouse Plant height phenotype of the transgenic poplar, 1bar=20cm; (c) Diameters of the top, middle and bottom of the transgenic poplar grown normally in the greenhouse for 13 weeks, 1bar=0.5cm.

Fig. 9 is a yield statistics chart of PtCYP85A3 transgenic poplar seedlings; in the figure, plant height, diameter, internode number, internode length, stem fresh weight, leaf fresh weight, leaf dry weight, leaf length and leaf width are shown in order in the figure, n≥6, error=±SD, P<0.01 is extremely significant (**), P<0.05 is significant (*).

Fig. 10 is the field phenotype of PtCYP85A3 transgenic poplar. In the figure, (a) WT, L3, L5 and L8 plant height phenotype, bar=30cm; (b) WT, L3, L5 and L8 diameter at breast height (left) and ground diameter (right) phenotype, bar=10mm; ( c-e) Statistical results of plant height, diameter at breast height and ground diameter of WT, L3, L5 and L8, n=16, error=±SD, P<0.01 is extremely significant (**), P<0.05 is significant (*).

Figure 11 is a paraffin section of PtCYP85A3 transgenic poplar; in the figure, (a) cross-section of WT, L3, L5 and L8 stems, bar=1 μm; (b-c) statistics of xylem and phloem thickness of WT, L3, L5 and L8, n≥4, error=±SD, P<0.01 is extremely significant (**), P<0.05 is significant (*).

Figure 12 is the transmission electron microscope results and lignocellulose content determination of PtCYP85A3 transgenic poplar; among the figure, (a) transmission electron microscope of WT, L3, L5 and L8, bar=5 μ m; (b-d) WT, L3, L5 and L8 cell wall Determination of dry matter, cellulose and lignin content; (e) Statistics of cell wall thickness of WT, L3, L5 and L8, n≥70, error=±SD, P<0.01 means extremely significant (**), P<0.05 means Significantly (*).

Fig. 13 is the salt resistance test of PtCYP85A3 transgenic poplar in the field; in the figure, the middle is wild-type WT, and the left and right are L3 and L8 respectively.

Figure 14 is the drought resistance test of PtCYP85A3 transgenic poplars in the field; in the figure, the wild type WT is in the middle, and L3 and L8 are on the left and right, respectively.

detailed description

The present invention will be described in detail through specific examples below. These embodiments are provided for a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

"Includes" or "comprises" mentioned throughout the specification and claims is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred implementation mode for implementing the present invention, but the description is for the purpose of the general principles of the specification, and is not intended to limit the scope of the present invention. The scope of protection of the present invention should be defined by the appended claims.

1. Materials and methods:

1. Medium

MS ₀ medium: 4.43g/L MS salts, 20g/L sucrose, pH adjusted to 5.8 with NaOH;

LB medium: 10g/L peptone (Typtone), 5g/L yeast extract (Yeast Extract), 10g/L sodium chloride, 0.8% agar powder added to solid medium;

2. Vector construction

Primers were designed to amplify the full-length coding sequence of PtCYP85A3 to obtain a DNA fragment of about 1395 bp, which was then ligated into the SmaI-digested cloning vector pBlueScript II KS (pKS; Stratagene), identified by enzyme digestion, and the sequenced correct plasmid was used XbaI and PstI were digested, and the digested fragment was recovered and ligated into the pCAMBIA1301/pCAMBIA2301 vector, in which PtCYP85A3 was driven by the CaMV 35S (Cauliflower mosaic virus) promoter. The primers used to construct the vectors are listed in Table 1.

Table 1

3. Recovery and Ligation of Nucleic Acid Fragments

The recovery and connection of nucleic acid fragments were carried out according to the corresponding kit instructions:

(1) Under ultraviolet light, cut out the gel block containing the desired DNA band and place it in a 1.5mL Eppendorf tube; add an appropriate amount of sol solution and incubate at 55°C for 10 minutes to fully dissolve the gel in the solution;

(2) Pour the solution into the adsorption column, place it at room temperature for 2min, centrifuge at 10000rpm for 30s, and pour off the liquid in the collection tube;

(3) Add 500 μL of rinse solution to the adsorption column, centrifuge at 10,000 rpm for 30 s, discard the liquid in the collection tube, and repeat once; centrifuge the empty tube at 10,000 rpm for 2 min;

(4) Take 30 μL of distilled water preheated at 65°C and place it on the center of the adsorption column, and place it at room temperature for 2 minutes;

(5) Put the adsorption column in a 1.5mL Eppendorf tube, centrifuge at 10000rpm for 2min, and the collected liquid contains the recovered target fragment;

(6) Take 7 μL of recovered nucleic acid fragments (with sticky ends after digestion) and 1 μL of corresponding plasmid vectors (with the same sticky ends after digestion) into a 1.5 mL Eppendorf tube, add 1 μL of T4DNA Ligase, 1 μL of 10×T4 DNA Ligase buffer, ligate overnight at 16°C;

(7) The ligation product can be directly used to transform T1 or DH5α strains.

4. Transformation of E. coli

(1) Thaw DH5α or T1 competent cells in ice (or at room temperature for a while, insert them into ice quickly when the cells are in a state of ice-water mixing), add the target DNA (plasmid or ligation product) and mix gently, statically on ice set for 30min;

(2) Heat shock in a water bath at 42°C for 90s, quickly put it back on ice and let it stand for 2min;

(3) Add 900 μL of sterile LB medium without antibiotics to the centrifuge tube, mix well and resuscitate at 37°C, 200rpm for 60min;

(4) Collect bacteria by centrifugation at 5000rpm for 1min, take about 100μL of supernatant and gently pipet the resuspended bacteria block (if it is blue-white screening, add 50μL X-Gal, 10μL IPTG), mix and spread until the bacteria containing the corresponding antibiotics On LB medium;

(5) Place the plate upside down and place it in a 37°C incubator for overnight culture.

5. Transformation of Agrobacterium

(1) Take the competent Agrobacterium stored at -80°C and thaw on ice. Add 1 μg (volume not more than 10 μL) plasmid DNA to every 100 μL of competent cells, mix well, let stand on ice for 30 minutes, and liquid nitrogen for 1 minute;

(2) Heat shock in a water bath at 37°C for 5 minutes, quickly put it back on ice and let it stand for 5 minutes;

(3) Add 900 μL of sterile LB medium without antibiotics, shake and culture at 28°C for 2-3h;

(4) Collect the bacteria by centrifuging at 6000rpm for 1min, take about 100μL of the supernatant and gently pipette the resuspended bacteria, spread it on the LB plate containing the corresponding antibiotic, and incubate it upside down in a 28°C incubator for 2-3 days.

6. Mini-extraction of plasmids

(1) Pour 2mL of the bacterial solution into an Eppendorf tube and centrifuge at 12000rpm for 1min;

(2) Discard the supernatant, and suspend the pellet in 300 μL Solution I (can be properly lysed for a few minutes); add 300 μL Solution II, and gently invert to mix (place at room temperature for 5 minutes); add 300 μL Solution III, and gently invert to mix;

(3) Centrifuge at 12000rpm for 10min, carefully absorb the supernatant; add an equal volume of isopropanol to the supernatant, and precipitate at -20°C for 20min;

(4) Centrifuge at 12000rpm for 10min; discard the supernatant, add 1mL of 75% ethanol to the precipitate to wash; centrifuge at 12000rpm for 2min, discard the supernatant; dry the precipitate at 37°C or room temperature;

(5) Add 30 μL sterile water to each tube to dissolve the plasmid;

(6) 0.8% agarose electrophoresis to detect the plasmid.

The plasmid extract composition is as follows:

Solution I (Resuspension solution): 50mM Tris-HCl (pH 7.5), 10mM EDTA (pH 8.0), RNase A 100μg/mL

Solution II (Lysis solution): 0.2M NaOH, 1% SDS

Solution III (Neutralization solution): 1.32M KAc (pH 4.8)

7. Enzyme digestion identification and electrophoresis of the plasmid

(1) In a 10 μL reaction system, add 5 μL extracted plasmid DNA (100-200 ng/μL), 1 μL of the corresponding digestion buffer, 0.5 μL (0.5 U) of enzyme, make up the volume to 10 μL with ddH ₂ O, 37°C Enzyme digestion for 2-3h;

(2) After enzyme digestion, add 1 μL 10×Loading Buffer to the reaction system and mix well, electrophoresis is completed in agarose gel, and detection is performed under ultraviolet light.

8. Minimized extraction of plant genomic DNA

(1) Add 400 μL of DNA extraction buffer and a steel ball of appropriate size to a 1.5 mL sterile centrifuge tube;

(2) Take a piece of leaf tissue with scissors, put it into a centrifuge tube, and crush the sample with a grinder (60Hz, 60s);

(3) Centrifuge at 10000rpm for 10min, take 300μL supernatant and transfer to another new centrifuge tube;

(4) Add an equal volume (300 μL) of isopropanol, precipitate for 10 minutes, and then centrifuge at 10,000 rpm for 10 minutes;

(6) Discard the supernatant, add 1 mL of 75% ethanol to the precipitate and wash once; then centrifuge at 10,000 rpm for 5 min, discard the supernatant, and hang upside down to dry at room temperature;

(7) Add 50-100 μL H ₂ O to dissolve for more than 10 minutes, then centrifuge at 5000 rpm for 1 minute, and collect the supernatant.

The composition of 100mL DNA extraction solution is as follows: 20mL 1mol/L Tris-HCl(pH 7.5)+250mM NaCl+5mL0.5mol/L EDTA(pH 8.0)+0.5%SDS+75mL H ₂ O

9. Extraction of plant total RNA

(1) Take an appropriate amount of fresh plant material, grind it into powder with liquid nitrogen, and transfer it to a 1.5mL Eppendorf tube;

(2) Add 1mL RNAiso Reagent, mix well, and let stand at room temperature for 10min;

(3) Add 1/5 volume of chloroform, shake and mix well, let stand at room temperature for 10 minutes; then centrifuge at 4°C, 12000rpm for 15 minutes;

(4) Transfer the upper liquid to a new Eppendorf, add an equal volume of isopropanol, let stand at 4°C for 10 minutes; then centrifuge at 12,000 rpm for 10 minutes at 4°C;

(5) Discard the supernatant, add 1 mL of 75% ethanol to the precipitate to clean the precipitate, centrifuge at 12000 rpm for 5 min;

(6) After the RNA precipitation was dried at room temperature, 30 μL of DEPC water was added to dissolve it, and the total RNA was analyzed by 2% denatured agarose gel electrophoresis, and stored at -20°C.

10. Reverse Transcription of Total RNA

PolyA mRNA first-strand reverse transcription using The reaction system of II Reverse Transcriptase (Weizan Biology, Nanjing) is shown in Table 2:

Table 2

Pipette gently to mix, 10min at 25°C, 30min at 50°C, 5min at 85°C, and then quickly placed on ice.

The reverse transcription product can be stored at -20°C for a short period of time, do not freeze and thaw repeatedly. The reverse transcription product (or after appropriate dilution) can be directly used for PCR detection.

11. PCR, RT-PCR and Real-time PCR

The PCR reaction system is shown in Table 3:

table 3

The PCR amplification program is shown in Table 4:

Table 4

The Real-time PCR reaction system is shown in Table 5:

table 5

Real-time PCR three-step amplification program is shown in Table 6:

Table 6

12. Protoplast transformation of poplar leaves and observation of subcellular localization of fluorescent proteins

(1) Pipette 15mL of the enzymatic hydrolysis solution into a petri dish, stick the leaves (about 40 pieces) of poplar trees with 3M black tape to remove the lower epidermis, place them in the enzymatic hydrolysis solution, and ensure that they are submerged in the enzymatic hydrolysis solution. -4h, shake gently at 40rpm, take part of the green liquid for microscopic examination;

(2) Filter the mixed liquid after enzymolysis with a 100-200 mesh sieve, and collect the filtered green liquid with a 15mL round-bottomed centrifuge tube;

(3) Centrifuge at 100g for 15min at 4°C, with a brake of 3;

(5) Gently suck off the supernatant, add 4 mL of pre-cooled W5 solution to resuspend, centrifuge at 100 g for 1 min at 4 °C, and brake at 3;

(6) Gently suck off the supernatant, add 4 mL of pre-cooled W5 solution to resuspend, and let stand on ice for 30 minutes;

(7) Take a small amount of protoplast suspension for counting with a hemocytometer, and dilute with an appropriate amount of W5 solution to a cell concentration of 2.5×10 ⁵ cells/mL;

(8) At room temperature, centrifuge at 100g for 1min with a brake of 3, gently suck off the supernatant, and resuspend with an appropriate amount of MMG solution;

(9) Add 10 μL of plasmid (10-20 μg) to a 2 mL centrifuge tube;

(10) Add 100 μL of protoplasts and mix gently;

(11) Add 110 μL PEG solution and mix gently;

(12) Place at 23°C for 30 minutes;

(13) First add 220 μL of W5 solution, mix slowly, then add 440 μL of W5 solution and mix, and finally add 880 μL of W5, mix well, and centrifuge at 100 g for 1 min at room temperature;

(14) Gently suck off the supernatant, resuspend with 1mL W5 solution, keep in the dark at 23°C for 6-18h;

(15) Take a small amount of liquid and observe under a fluorescent confocal microscope (Zeiss LSM 510META).

Enzymolysis solution: 1-1.5% cellulase R-10, 0.2-0.4% macerozyme R-10, 0.4M mannitol, 20mM KCl, 20mM MES (pH 5.7), 55°C water bath for 10min, add 10mM CaCl ₂ after cooling to room temperature, 5 mM β-mercaptoethanol, 0.1% BSA

PEG solution (40%, v/v): 1 g PEG 4000 (Fluka, #81240), 750 μL H ₂ O, 625 μL 0.8M mannitol, 250 μL 1M CaCl ₂

W5 solution: 154mM NaCl, 125mM CaCl2, 5mM KCl, 5mM Glucose, 0.03% MES, adjust pH to 5.7 with KOH, high temperature sterilization

MMG solution: 0.4M mannitol, 15mM MgCl ₂ , 0.1% MES, adjust pH to 5.6 with KOH, high temperature sterilization

13. Genetic transformation of poplar (Wang et al., 2011)

(1) Inoculate 50 mL of LB liquid medium (containing Rif 100 μg/mL, Kan 50 μg/mL) containing the EHA105 strain to be transformed into the vector, and culture on a shaker at 28°C until OD ₆₀₀ =0.5-0.6;

(2) In the ultra-clean workbench, transfer the working bacteria solution into a centrifuge tube, centrifuge at 6500 rpm for 5 minutes, and dilute the collected bacteria with liquid MS ₀ culture solution to a final concentration of OD ₆₀₀ =0.2-0.4;

(3) Take the leaves of sterile poplar tissue culture seedlings, remove the main veins, cut into small pieces with a size of about 1cm×1cm, soak them in the Agrobacterium suspension diluted with MS ₀ for 5-20min, and shake gently during the period Several times or placed in a low-speed shaker;

(4) Take out the soaked leaves and place them on sterile filter paper, blot the bacterial solution on the surface, transfer to the co-cultivation medium, and co-cultivate for 48 hours at 25°C in the dark;

(5) Wash the leaves three times with sterile water, transfer to the differentiation medium, and cultivate at 24°C;

(6) After one week, the explants are transferred to the screening culture, and the fresh medium is replaced once in 7-10 days;

(6) Screening and culturing for about 30 days, the transformed cells of the leaf explants differentiate into resistant buds, and when they grow to 5-6 leaves, they are transferred to the rooting medium to take root.

Co-cultivation medium: MS ₀ basic medium + 0.01mg/L TDZ + 0.2mM/L AS

Differentiation medium: MS ₀ basic medium + 0.5mg/L6-BA + 0.1mg/L NAA + 0.01mg/L TDZ + 400mg/LTim

Screening medium: MS ₀ basic medium + 0.1mg/L NAA + 400mg/L Tim + 10mg/L Hyg

Rooting medium: MS ₀ basic medium + 0.1mg/L NAA + 10mg/L Hyg

14. Genetic transformation of tomato (Zhang et al., 2001)

(1) Surface sterilize the tomato seeds with 10% sodium hypochlorite for 5 minutes, wash them with sterile water for 3-5 times, then put the seeds on sterile filter paper to dry, and sow them on the MS ₀ medium;

(2) Inoculate 30 mL of LB liquid medium (containing Rif 100 μg/mL, Kan 50 μg/mL) containing the EHA105 strain to be transformed into the vector, and cultivate at 28°C until OD ₆₀₀ =0.6-0.8;

(3) In the ultra-clean workbench, transfer the working bacteria solution into a centrifuge tube, centrifuge at 6000 rpm for 5 minutes, and dilute the collected bacteria with liquid MS ₀ culture solution to a final concentration of OD ₆₀₀ =0.3-0.5;

(4) Cut off the hypocotyl of the sterile tomato sown for one week, split the cotyledon from the middle, then cut the cotyledon into three sections, put it into the Agrobacterium suspension diluted by MS ₀ and soak it for 3-5min, during which it can lightly shake a few times;

(5) Take out the soaked leaves and place them on sterile filter paper, blot dry the bacterial solution adsorbed on the surface, transfer to the co-cultivation medium, and co-cultivate for 48 hours at 25°C in the dark;

(6) The material was washed three times with sterile water, transferred to the screening medium, cultivated at 24°C, and replaced with a fresh medium every 10 days;

(7) After screening and culturing for 2 months, the cotyledon explants differentiate into resistant seedlings, and when the aerial part grows to 2-4 cm, the differentiated seedlings are cut off from the explants and transferred to the rooting medium for rooting.

(8) After 2-4 weeks, samples were taken for PCR detection and GUS staining;

(9) Transplant the transgenic positive seedlings into flower pots (black soil: vermiculite: perlite=3:1:1), and put them in the greenhouse for cultivation, day temperature is 25°C, night temperature is 15-20°C, and light conditions ( 16h light/8h dark), water every three days, and water every other week for flowers.

Co-cultivation medium: MS ₀ basic medium + 1.0mg/L ZT + 0.1mg/L IAA + 0.1mM/LAS

Screening medium: MS ₀ basic medium+1.0mg/L ZT+0.1mg/L IAA+400mg/L Tim+50mg/LKan

Rooting medium: MS ₀ basic medium + 0.1mg/L IAA + 50mg/L Kan

15. GUS histochemical analysis

The plant samples were taken and put into GUS reaction buffer, and kept at 37°C. When the color intensity is sufficient, suck out the GUS reaction buffer, add 75% ethanol to decolorize, and replace the ethanol every few hours. 50% glycerin was used to prevent water from evaporating too quickly and causing the material to shrink.

GUS reaction buffer: 100 mM NaH ₂ PO ₄ , 10 mM EDTA, 0.5 mM K ₃ [Fe(CN) ₆ ], 0.5 mM K ₄ [Fe(CN) ₆ ], 0.1% Triton X-100, adjust pH to 7.0, add X-Gluc before use to a final concentration of 0.2-0.5mg/mL

16 Basic techniques for making paraffin sections

The paraffin section method includes the steps of taking material, fixing, washing and dehydrating, clearing, dipping in wax, embedding, slicing and sticking, dewaxing, staining, dehydrating, clearing, and sealing.

16.1 Material collection

Select the poplar material of the same part according to the requirements. The collected specimens should pay attention to moisturizing. After rinsing, cut out samples of appropriate size (the smaller the better, the volume generally does not exceed 0.5cm) and quickly put them into the fixative solution.

16.2 Fixed

FAA fixative (50% or 70% ethanol 90mL + glacial acetic acid 5mL + 37-40% formaldehyde 5mL) was fixed for 24h or longer, and the sample was vacuumed to sink to the bottom. The amount of fixative used is usually about 20 times that of the material block, and the fixation time depends on the size, thickness, and penetration speed of the fixative of the material block.

16.3 Dehydration, clearing and waxing

70% absolute ethanol (2h)→85% absolute ethanol (2h)→95% absolute ethanol (2h)→100% absolute ethanol (2h)→1/2 absolute ethanol+1/2xylene (2h )→xylene (2h)→1/2xylene+1/2 crushed wax (in an incubator at 36°C overnight, add a small amount of safranin)→soak in wax (raise the temperature to 56°C the next morning, open the lid to make it transparent The agent evaporates, and the paraffin solution is poured after 2 hours. The plant material is moved into a small beaker of molten pure paraffin with a high melting point, and the molten pure paraffin is changed again in 2-4 hours, and the embedding can be carried out in 2-4 hours);

Main items:

(1) Gradually dehydrate for about 2 hours. The dehydration time can be appropriately shortened for small and tender materials, and the dehydration time for large and hard materials (high degree of lignification) can be longer, but if the dehydration time is too long in high-concentration dehydrating agents, it will cause The material becomes brittle, which is not conducive to slicing.

(2) The consumption of dehydrated ethanol is 3-5 times of the material volume.

16.4 Embedding, Sectioning and Adhesive

(1) Fold and embed the carton with kraft paper or hard, smooth paper;

(2) Material embedding: Put molten paraffin into the carton, arrange the material with tweezers or dissecting needles heated on an alcohol lamp (quickly, to avoid paraffin crystallization), and then put the embedding carton into cold water cooling and solidification;

(3) Wax repair: pull one material for each piece, and cut it with double-sided blades;

(4) Fast wax sticking: 2×2×2.5-3cm rectangular wooden block, with a grid carved on one end of the long axis; one end of the wooden block with a grid is immersed in melted waste paraffin, and the wax block is repaired into a table with a wide bottom and a narrow top. For the body, use a hot dissecting needle to paste the wide surface of the wax block on one side and the wax surface of the wooden block on the other side. After pasting, the small wax block is reinforced with hot dissecting needles around it so that no gaps are left. The cut surface should be parallel to the knife edge;

(5) Slicing: brush support (the thickness depends on the material and needs, and the wax tape is cut according to requirements);

(6) Adhesive slices: clean the slides → drip distilled water (gelatin is not required) → place the slices on the liquid surface with a dissecting needle or knife → place them on the temperature platform → adjust the slices → absorb excess water with absorbent paper → warm After drying on the bench, place it in a 30°C incubator for 24 hours.

Precautions:

(1) Only by rapid cooling can the crystallization particles of the wax be smaller, and slices are used;

(2) Wax with a lower melting point (such as 54°C) is used for slices in winter, and wax with a higher melting point (such as 58-60°C) is used in summer, but paraffin with a low melting point is easy to flatten, and paraffin with a high melting point is not easy to flatten;

(3) The slides should be soaked in polylysine (diluted with mother liquor 10×) for 5 minutes in advance, and dried overnight.

16.5 Section dewaxing, staining, dehydration and clearing

Dried slices → xylene (5-10min) → xylene (5-10min) → 1/2 absolute ethanol + 1/2 xylene (5-10min) → absolute ethanol (5-10min) → 95% Absolute ethanol (5-10min)→85% absolute ethanol (5-10min)→70% absolute ethanol (5-10min)→50% absolute ethanol (5-10min)→distilled water→aniline blue (2-5min ) → washed twice with distilled water, and then observed under a microscope.

17 Determination of lignin and cellulose content

17.1 Isolation of cell walls

(1) Weigh 60-70mg of fresh or frozen material into a 2mL centrifuge tube;

(2) Add 1.5mL of 70% ethanol, vortex and mix;

(3) Centrifuge at 10000rpm for 10min;

(4) Discard the supernatant, add 1.5mL chloroform/methanol (1:1, v/v), mix and resuspend;

(5) centrifuge at 10000rpm for 10min, discard the supernatant;

(6) Add 500 μL of acetone to resuspend, and store at 35°C until the acetone evaporates (dried samples can be stored at room temperature until further processing);

(7) Add 1.5mL of 0.1M sodium acetate with pH 5.0 to resuspend the sample;

(8) Cap the centrifuge tube tightly and put it in a water bath at 80°C for 20 minutes;

(9) Cool on ice, add 35 μL 0.01% NaN ₃ , 35 μL amylase (amylase, 50 μL/mL H ₂ O; from Bacillus species, SIGMA), 17 μL pullulanase (pullulanase, 18.7 units from bacillus acidopullulyticus, SIGMA) in sequence , cap the centrifuge tube tightly, and vortex to mix;

(10) Overnight in a water bath at 37°C;

(11) 100°C water bath for 10 minutes to terminate the reaction;

(12) centrifuge at 10000rpm for 10min, discard the supernatant;

(13) Add 1.5 mL of distilled water, vortex to mix, centrifuge, discard supernatant, repeat three times;

(14) Add 500 μL of acetone to resuspend, and store at 35°C until the acetone evaporates (the dried sample is the isolated cell wall, which can be stored at room temperature until further processing).

17.2 Determination of cellulose content (refer to Foster et al., 2010a)

(1) Weigh 2 mg of the above-mentioned treated cell wall material in a 2 mL centrifuge tube;

(2) Add 1 mL of Updegraffreagent (acetic acid: nitric acid: water = 8:1:2, v/v);

(3) Cap the centrifuge tube tightly, vortex and mix well, and bathe in 100°C water for 30 minutes (to dissolve insoluble substances except crystalline cellulose);

(4) Cool on ice to room temperature, centrifuge at 10,000rpm for 15min, and discard the supernatant (150μL supernatant can be kept to prevent pouring out the sample);

(5) Add 1.5mL of distilled water, vortex to mix, centrifuge, discard the supernatant, repeat three times;

(6) Add 1.5 mL of acetone, vortex to mix, centrifuge, discard supernatant, repeat three times;

(7) Dry at 35°C until the acetone evaporates;

(8) Add 175 μL of 72% sulfuric acid (to completely dissolve the crystalline cellulose into glucose);

(9) Place at room temperature for 30 minutes, vortex and then rest for 15 minutes;

(10) Add 825 μL H ₂ O, vortex and mix;

(11) Centrifuge at 10000rpm for 5min (there may still be some brown insoluble lignin at the bottom of the tube);

(12) Utilize the anthrone colorimetric method to determine the glucose content in the supernatant, draw a standard curve with 1mg/mL glucose mother solution (stored at zero degrees), and prepare repeated standard samples of 0, 2, 4, 6, 8, 10 μg, That is, add 0, 2, 4, 6, 8, and 10 μL of mother solution respectively, and make up to 100 μL with water;

(13) Take 100 μL supernatant for each sample, add 900 μL H ₂ O, mix well, take 100 μL and add it to a 96-well polyethylene microtiter plate, and repeat three times for each group;

(14) Add 200 μL of freshly prepared anthrone solution (2 mg anthrone/mL concentrated sulfuric acid);

(15) Place in an oven at 80°C for 30 minutes (the sample turns from yellow to blue-green);

(16) Take out and cool to room temperature, mix;

(17) Use a microplate reader to read the absorbance value at 625nm;

(18) Use the glucose standard curve to calculate the glucose content of each sample, that is, the cellulose content in the sample.

17.3 Determination of lignin content (refer to Foster et al., 2010b)

(1) Weigh 1-1.5 mg of the above-mentioned treated cell wall material into a 2 mL centrifuge tube, leaving an empty tube as a control;

(2) Add 250 μL of acetone, collect the cell wall material at the bottom of the tube, and dry naturally until the acetone evaporates;

(3) Carefully add 100 μL of freshly prepared bromoacetyl solution (25% v/v acetylbromide in glacial acetic acid) along the tube wall;

(4) Cover the tube cap and put it in a water bath at 50°C for 2 hours;

(5) Water bath at 50°C for 1 hour, and vortex every 15 minutes;

(6) cooling to room temperature on ice;

(7) Add 400 μL 2M sodium hydroxide and 70 μL freshly prepared 0.5M hydroxylamine hydrochloride (hydroxylaminehydrochloride) in sequence, and vortex to mix;

(8) Make up to 2 mL with glacial acetic acid, and vortex to mix;

(9) Take 200 μL in a 96-well UV microburette, and use a microplate reader to read the absorbance at 280 nm;

(10) Utilize the following formula to calculate the lignin contained in the solution:

Wherein, abs represents the absorbance value, coefficient (Poplar=18.21; Grasses=17.75; Arabidopsis=15.69).

2. Test steps

1. PtCYP85A3 gene expression analysis: The PtCYP85A3 gene was cloned from the sequenced poplar species (Pulus trichocarpa) by bioinformatics analysis. Extract the total RNA of roots, stems, leaves and other different tissues of Populus trichocarpa and stems of different development stages, as well as different parts of the stem (epidermal and xylem), and analyze the tissue expression characteristics of PtCYP85A3 in Populus trichocarpa by RT-PCR technology; at the same time The total RNA of Populus trichocarpa seedlings treated with different concentrations of BRs was used to analyze the response of PtCYP85A3 to BRs.

2. Subcellular localization of PtCYP85A3 protein: Through the colocalization experiment of the fluorescent protein PtCYP85A3-YFP-labeled fusion protein and the marker fusion protein (ER-CFP) localized in the endoplasmic reticulum, the poplar mesophyll cell protoplasts were transiently transformed, To study the localization of PtCYP85A3 in poplar cells.

3. PtCYP85A3 function analysis: PtCYP85A3 was used to complement the CYP85A mutants of Arabidopsis thaliana (cyp85a2-2) and tomato (d ^x ), respectively; and the overexpression vector of PtCYP85A3 was used to transform Arabidopsis thaliana, and the phenotype of the transgenic Arabidopsis was analyzed; These experiments are used to confirm whether the PtCYP85A3 gene has similar biological functions to herbaceous plants.

4. Construct a gene expression vector that can be expressed in large quantities in crops, place the target gene PtCYP85A3 under the strong constitutive expression promoter 2×CaMV35S promoter, and construct Hyg and Kanamycin with hygromycin resistance genes Binary vector of the nptin resistance gene NPTII.

5. Introduce the above-mentioned gene expression vector into crops (tomatoes are kanamycin-resistant, poplars are hygromycin-resistant) by means of Agrobacterium-mediated methods, and under the drive of the promoter, the target gene is transgenic. It is expressed in large quantities in crops; and the transgenic tomato plants with kanamycin resistance and the transgenic poplar plants with hygromycin resistance gene are screened according to the selection marker gene on the gene expression vector.

6. Perform PCR on resistant plants to detect whether the exogenous gene is integrated into the genome; perform PCR, GUS staining, RT-PCR and real-time PCR on the homozygous lines for the target gene to detect the expression level of the exogenous gene.

7. The effect of PtCYP85A3 on the growth and development of tomato and poplar: the transgenic poplar with significant overexpression of PtCYP85A3 was moved to the soil for phenotypic analysis, and the changes in leaves, plant height, diameter, biomass and growth rate were observed; the phenotype was determined to be obvious The content of BRs (leaves) in transgenic poplars was used to study the effects of changes in the content of endogenous BRs on the growth and development of poplars.

8. The effect of PtCYP85A3 on the development of poplar stems: the changes of phloem, cambium and xylem in transgenic poplar stems were studied by tissue section method; the cell wall components (cellulose, lignin and various cell wall sugars) in transgenic poplar were determined The change of content; the change of fiber cell wall thickness was observed by transmission electron microscope; these experiments were used to study the influence of BRs on the secondary development of poplar.

9. Field test of transgenic poplar: For transgenic poplar with significantly improved biomass, growth speed, or stress resistance, apply to the State Forestry Administration for a pilot test of transgenic poplar, and conduct field tests in the natural state of the field or in saline-alkali land to detect the transgenic Stability of poplar traits.

3. Test results

1. The comparison results show that PtCYP85A3 has a high similarity with Arabidopsis, rice, tomato and other CYP85A, among which the similarity with AtCYP85A2 is as high as 68.52%. Phylogenetic tree analysis shows that PtCYP85A3 and AtCYP85A2 are in the same branch (Figure 1) . Through homologous sequence alignment, we found three genes homologous to tomato DWARF in poplar, which were named PtCYP85A1 (XP_002305393), PtCYP85A3 (XP_002330918) and PtCYP85A4 (XP_002316079). The amino acid sequence alignment is shown in Figure 1a . Genebank序列号如下：Arabidopsis thalianaAtCYP85A1(BAB60858.1)和AtCYP85A2(BAC55065.1)，Pisum sativum PsCYP85A1(BAF56235.1)和PsCYP85A6(BAF56236.1)，lycopersicon esculentum LeCYP85A1(DWARF，AAB17070.1)和LeCYP85A3(BAD98244 .1), Oryza sativa OsCYP85A1 (BAC45000.1), Vitisvinifera VvCYP85A1 (ABB60086.1), Populus trichocarpa PtCYP85A1 (XP_002305393), PtCYP85A3 (XP_002330918) and PtCYP85A3 (6070918) Among them, six substrate recognition sites, SRS (sixsubstrate recognition sites), Pro-rich motif, a dioxygen binding domian, a Glu-X-X-Arg motif, and a Heme binding domain are very conserved.

Among them, the similarity between PtCYP85A3 and AtCYP85A2 was as high as 91.40%, and the similarity between PtCYP85A3 and LeCYP85A1 was as high as 94.18%. PtCYP85A3 has a relatively conserved domain of cytochrome P450 oxidase: Pro-rich motif, adioxygen binding domian, a Glu-X-X-Arg motif, a Heme binding domain.

The phylogenetic tree is shown in Figure 1b. PtCYP85A3 is in the same branch as AtCYP85A2 and LeCYP85A1, and has a high genetic relationship. The full length of the PtCYP85A3 gene is 3274bp, which contains 9 exons and 8 introns. The length of the region is 1395bp, encoding a protein of 464 amino acids, hydrophobic amino acids account for 43.7%, the molecular weight of the protein is 53.65kD, and the isoelectric point is 9.46.

2. Extract the total RNA from each tissue of Populus trichocarpa growing for three months, and analyze the distribution of PtCYP85A3 transcripts in different tissue parts of Populus by RT-PCR and Real-time PCR. The results showed that PtCYP85A3 was expressed in various tissues such as terminal buds, young leaves, mature leaves, petioles, phloem and xylem of elongated stems, phloem and xylem of thickened stems, and roots, and the expression level in young tissues Higher, especially the highest expression level in young leaves (Figure 2).

3. Extract the total RNA of poplar tissue culture seedlings treated with 100nM BL for different times and different concentrations for 30 minutes, and analyze the relative expression of PtCYP85A3 by RT-PCR and Real-time PCR after reverse transcription . The results showed that the expression level of PtCYP85A3 showed a significant downward trend with the increase of treatment time and concentration (Fig. 3), which indicated that PtCYP85A3 was negatively regulated by BRs in Populus trichocarpa.

4. Construct the PtCYP85A3-YFP fusion vector, use pA7-YFP as a blank control, and use ER-YFP (Nelson et al., 2007) as a positive control for endoplasmic reticulum localization, use the PEG method to transform the protoplasts of poplar mesophyll cells, and fuse The protein is transiently expressed under the drive of the constitutive 35S promoter, and its subcellular localization can be observed under confocal laser microscopy. The transient expression system of tobacco epidermal cells and poplar protoplasts showed that PtCYP85A3 was localized in the endoplasmic reticulum (Figure 4).

5. PtCYP85A3 can partially complement the tomato (d ^x ) mutant (Figure 5).

6. The PtCYP85A3 gene was constructed on a plant expression vector, and transformed into tomato Micro-TOM by Agrobacterium tumefaciens, and four independent transgenic lines were obtained. The molecular identification results are shown in Figure 6.

7. Overexpression of PtCYP85A3 can increase the yield and biomass of transgenic tomato.

The statistical results of overexpression of PtCYP85A3 on tomato biomass and yield are shown in Table 7.

Table 7 Effect of overexpression of PtCYP85A3 on biomass and yield of tomato

The plant height was measured at two months, and the number of flowers, the length of the first internode, the length of the second internode and the length of petiole were counted in the early stage of reproductive growth; the fresh weight on the ground, the number of fruits, the yield per plant and the freshness of single fruit were counted at three months. Heavy. n=6, error=±SD, P<0.01 is extremely significant (**), P<0.05 is significant (*). From the statistical results, overexpression of PtCYP85A3 can promote the biomass and yield of tomato.

8. The PtCYP85A3 gene was constructed on a plant expression vector, and transformed into poplar by Agrobacterium tumefaciens, and eight independent transgenic lines were obtained. PCR molecular identification proved that these genes were integrated in the poplar genome, and RT-PCR analysis proved that the PtCYP85A3 gene was overexpressed at the RNA level in multiple transgenic lines ( FIG. 7 ).

9. The phenotypes of various indexes of poplar trees potted in artificial climate chamber.

See Figure 8 for plant height and diameter of potted poplar in artificial climate chamber, see Table 8 for biomass statistics, and all the data in Table 8 are the measurement results of poplar grown for 13 weeks, and the diameter and internode length are all measured in the middle As a result, the leaves with long leaves and wide petioles are all mature unfolded leaves. n=6, error=±SD, P<0.01 is extremely significant (**), P<0.05 is significant (*). The results showed that overexpression of PtCYP85A3 could increase the biomass of poplar.

Table 8 Effect of overexpression of PtCYP85A3 on the biomass of poplar

Note: In the table, the unit of plant height, leaf length, leaf width and petiole is cm, the unit of diameter and internode length is mm, and the unit of stem fresh weight is g.

According to the RT-PCR results, the L5 strain with the highest expression level and the L3 and L8 strains with medium expression levels were selected from the eight transgenic lines, and a batch was retransplanted into the greenhouse. When the normal growth reached 13 weeks, the statistics Various biomass indexes such as plant height were analyzed (Fig. 9).

10. The overexpression of PtCYP85A3 in poplar can increase the content of BRs in transgenic poplar.

Three-month-old young poplar leaves (because PtCYP85A3 has the highest expression level in young leaves) were taken respectively, and three independent individual plants were randomly selected from each strain, then ground into powder with liquid nitrogen, and mailed to Wuhan Lv Jianke Ruixin Technology Co., Ltd. commissioned a third party to determine the content of brassinosteroids. The results are shown in Table 9. From the statistical results, it can be seen that the overexpression of PtCYP85A3 in poplar can increase the content of BRs in transgenic poplar.

Table 9 Determination results of BRs content in PtCYP85A3 transgenic poplar

11. PtCYP85A3 gene can significantly increase the biomass of poplar.

The field phenotype of the PtCYP85A3 transgenic poplar is shown in Figure 10. The phenotype of the biomass in Figure 10 proves that the overexpression of the PtCYP85A3 gene in the poplar can increase the plant height and stem diameter of the poplar, and the paraffin section of the PtCYP85A3 transgenic poplar (Figure 11) Based on the transmission electron microscope results and lignocellulose content determination results of PtCYP85A3 transgenic poplar ( FIG. 12 ), it can be concluded that the PtCYP85A3 gene can significantly increase the biomass of poplar, especially the synthesis of xylem.

12. The wild type and two transgenic lines (L3 and L8) were planted in saline-alkali land and dry land respectively, and their salt tolerance and drought resistance were observed. It was found that the biomass of the transgenic poplar was still higher than that of the wild type under saline-alkali and drought conditions (Figure 13 and Figure 14).

Although the present invention has been described in detail with general descriptions and specific examples above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

SEQUENCE LISTING

<110> Ludong University

<120> A poplar PtCYP85A3 gene and its application

<130> 2017

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1553

<212>DNA

<213> Populus trichocarpa

<400> 1

agagcatttc gataacacta gcagctcttt ccgaggcggg cttggagctg tttttcttcc 60

ttggaagcat caatggcagt tctcttgatg gttcttgtgg cagttctctt tttgttctgt 120

atctcctctg ctttgttgag gttgaacgag gtgagatata ggaagaaagg gttgcctcca 180

ggtactatgg gatggccagt ctttggagag accactgagt ttctaaagca aggtccaaac 240

ttcatgaaga atcagagagc aaggtatggg agtattttca aatcccacat tctggggtgt 300

cctaccatg tgtccatgga tccagagctc aatcgataca tcctaatgaa cgagggaaag 360

ggccttgttc ctggttaccc tcagtccatg ctggatatct taggcaatcg caacattgca 420

gcagttcatg gctccactca caagtacatg agaggggcat tattatccct cattagcccc 480

accatgatca gagaacaact tttgccaaca attgatgagt tcatgagaac ccacctcagc 540

tactgggata ccaaaattat tgacattcaa caaatgacta aggagatggc acttctctct 600

gcacttaagc aaattgctgg cactgattcg tgctcaatat ctcaagcatt catgcctgag 660

tttttcaggc tggttttagg cactttgtca ttgccaattg accttcctgg cacaaattat 720

cgacaaggag tccaggcgag aaaaaatatt gtacgcatgt taaggcagct aatagacggg 780

aggagggcat cgaaattata ccaccaggac atgcttggtc gacttatgag aactgaagaa 840

aacaaattta aactaacaga tgaagagata attgatcaaa taatcacaat tttgtactct 900

ggctacgaaa cggtttcgac cacttcaatg atggcagtca agtatctgca tgatcaccca 960

agagttcttc aggagctaag aaaagagcat ttcgcaatta gagaaaagaa aaggcctgag 1020

gatccaatcg atttaaatga ccttaaatcg atgcgtttta ctcgtgccgt gatttttgag 1080

acctcaagat tggctacaat agtaaatggg gttttgagga agactactaa agaaatggaa 1140

ctaaatagat ttgtgattcc aaaaggatgg agaatctacg tttacacaag ggagataaac 1200

tatgatccat atttatatcc tgacccattc tcctttaacc catggagatg gctggacaaa 1260

agtttggagt ctcaaaacta tctcttcatt tttggaggag gtaccaggca gtgtccagga 1320

aaggagctag gaatagctga gatttcaact ttccttcatt attttgtaac tagatacaga 1380

tgggaagagg ttggaggaga ctcattaatg aaatttccaa gagttgaagc accaaatggg 1440

ctacacatta gggtctcatc tcactaacta ataataacct atgcatgtac agaagagaga 1500

gagatatagc atatcagagc tgataatgat gttctaacat gttagaaagt gaa 1553

<210> 2

<211> 464

<212> PRT

<213> Populus trichocarpa

<400> 2

Met Ala Val Leu Leu Met Val Leu Val Ala Val Leu Phe Leu Phe Cys

1 5 10 15

Ile Ser Ser Ala Leu Leu Arg Leu Asn Glu Val Arg Tyr Arg Lys Lys

20 25 30

Gly Leu Pro Pro Gly Thr Met Gly Trp Pro Val Phe Gly Glu Thr Thr

35 40 45

Glu Phe Leu Lys Gln Gly Pro Asn Phe Met Lys Asn Gln Arg Ala Arg

50 55 60

Tyr Gly Ser Ile Phe Lys Ser His Ile Leu Gly Cys Pro Thr Ile Val

65 70 75 80

Ser Met Asp Pro Glu Leu Asn Arg Tyr Ile Leu Met Asn Glu Gly Lys

85 90 95

Gly Leu Val Pro Gly Tyr Pro Gln Ser Met Leu Asp Ile Leu Gly Asn

100 105 110

Arg Asn Ile Ala Ala Val His Gly Ser Thr His Lys Tyr Met Arg Gly

115 120 125

Ala Leu Leu Ser Leu Ile Ser Pro Thr Met Ile Arg Glu Gln Leu Leu

130 135 140

Pro Thr Ile Asp Glu Phe Met Arg Thr His Leu Ser Tyr Trp Asp Thr

145 150 155 160

Lys Ile Ile Asp Ile Gln Gln Met Thr Lys Glu Met Ala Leu Leu Ser

165 170 175

Ala Leu Lys Gln Ile Ala Gly Thr Asp Ser Cys Ser Ile Ser Gln Ala

180 185 190

Phe Met Pro Glu Phe Phe Arg Leu Val Leu Gly Thr Leu Ser Leu Pro

195 200 205

Ile Asp Leu Pro Gly Thr Asn Tyr Arg Gln Gly Val Gln Ala Arg Lys

210 215 220

Asn Ile Val Arg Met Leu Arg Gln Leu Ile Asp Gly Arg Arg Ala Ser

225 230 235 240

Lys Leu Tyr His Gln Asp Met Leu Gly Arg Leu Met Arg Thr Glu Glu

245 250 255

Asn Lys Phe Lys Leu Thr Asp Glu Glu Ile Ile Asp Gln Ile Ile Thr

260 265 270

Ile Leu Tyr Ser Gly Tyr Glu Thr Val Ser Thr Thr Ser Met Met Ala

275 280 285

Val Lys Tyr Leu His Asp His Pro Arg Val Leu Gln Glu Leu Arg Lys

290 295 300

Glu His Phe Ala Ile Arg Glu Lys Lys Arg Pro Glu Asp Pro Ile Asp

305 310 315 320

Leu Asn Asp Leu Lys Ser Met Arg Phe Thr Arg Ala Val Ile Phe Glu

325 330 335

Thr Ser Arg Leu Ala Thr Ile Val Asn Gly Val Leu Arg Lys Thr Thr

340 345 350

Lys Glu Met Glu Leu Asn Arg Phe Val Ile Pro Lys Gly Trp Arg Ile

355 360 365

Tyr Val Tyr Thr Arg Glu Ile Asn Tyr Asp Pro Tyr Leu Tyr Pro Asp

370 375 380

Pro Phe Ser Phe Asn Pro Trp Arg Trp Leu Asp Lys Ser Leu Glu Ser

385 390 395 400

Gln Asn Tyr Leu Phe Ile Phe Gly Gly Gly Thr Arg Gln Cys Pro Gly

405 410 415

Lys Glu Leu Gly Ile Ala Glu Ile Ser Thr Phe Leu His Tyr Phe Val

420 425 430

Thr Arg Tyr Arg Trp Glu Glu Val Gly Gly Asp Ser Leu Met Lys Phe

435 440 445

Pro Arg Val Glu Ala Pro Asn Gly Leu His Ile Arg Val Ser Ser His

450 455 460

Claims (7)

1. A poplar PtCYP85A3 gene, characterized in that the gene is the nucleotide sequence shown in SEQ ID NO.1. 2. The protein encoded by the poplar PtCYP85A3 gene of claim 1, wherein the protein is the amino acid sequence shown in SEQID No.2. 3. The carrier containing the poplar PtCYP85A3 gene according to claim 1. 4. the application of poplar PtCYP85A3 gene described in claim 1 in improving plant fruit yield and enhancing plant stress resistance. 5. the application of the poplar PtCYP85A3 gene described in claim 1 in improving the wood biomass of woody plants. 6. The application according to claim 4, characterized in that the stress resistance includes salt tolerance and drought resistance. 7. The use according to claim 4, wherein said plants comprise tomato and poplar.